The green fluorescent protein (GFP) from the Pacific Northwest jellyfish, Aequorea Victoria, has been used extensively in molecular and cell biology as a fluorescent marker. It is a 238 amino acid protein that generates its own fluorescent chromophore. The spontaneous generation of the chromophore is achieved by cyclization of the internal Ser65-Tyr66-Gly67 sequence followed by oxidation of Tyr 66 in the presence of molecular oxygen (Heim et al., Proc. Natl. Acad. Sci. USA 91:12501-12504, 1994). The overall fold of the protein consists of an 11-stranded β-barrel capped by α-helices at both ends and contains a coaxial α-helix from which the chromophore is generated (Brejc et al., Proc. Natl. Acad. Sci. USA 94:2306-2311, 1997; Ormö et al., Science 273:1392-1395, 1996; Yang et al., Nat. Biotech. 14:1246-1251, 1996). GFP is unique among light emitting proteins, because it does not require the presence of any cofactors or substrates for the production of green light.
Wild-type GFP has absorption maxima at 398 and 475 nm (Morise et al., Biochemistry 13:2656-2662, 1974). Excitation at either of these wavelengths leads to emission of green light at 508 nm (Morise et al., 1974). The usefulness of GFP has been greatly enhanced by the availability of mutants with a broad range of absorption and emission maxima (Heim et al., Proc. Natl. Acad. Sci. USA 91:12501-12504, 1994; Ormö et al., Science 273:1392-1395, 1996). These mutants have made possible multicolor reporting of cellular processes by allowing for the simultaneous observation of two or more gene products labeled with different colored GFP variants (Rizzuto et al., Curr. Biol. 6:183-188, 1996). In addition, fluorescence resonance energy transfer (FRET) experiments using different colored GFP's have been used to study protein-protein interactions in vivo (Heim et al., Curr. Biol. 6:178-182, 1996; Mitra et al., Gene 173:13-17, 1996).
More recently, GFP variants have been shown to be sensitive to pH (Wachter et al., Biochemistry 36:9759-9765, 1997; Elsliger et al., Biochemistry 38:5296-5301, 1999). As a consequence, they have been used as noninvasive intracellular pH indicators. For instance, Kneen et al. employed the GFP mutant S65T/F64L to determine the pH of the cytoplasm of CHO and LLC-PK1 cell lines (Kneen et al., Biophys. J. 74:1591-1599, 1998). Since GFP is genetically encoded, it can be specifically targeted to various subcellular compartments, which is a task not possible with small molecule fluorescent dyes (Llopis et al., Proc. Natl. Acad. Sci. USA 95:6803-6808, 1998). Therefore, Llopis and co-workers used the GFP variant S65G/S72A/T302Y/H231L, which has an increased pKa, to measure the alkaline pH of mitochondria, golgi, and the cytosol of HeLa cells and rat neonatal cardiomyocytes (Llopis et al., 1998). These reports were the first to show that GFP variants could be used as biosensors and not just simple fluorescent markers. However, more recently GFP has been shown to be sensitive to halide ions and through a fusion with calmodulin, GFP's fluorescence can also vary in response to calcium ion concentration (Wachter et al., Curr. Biol. 9:R628-R629, 1999; Miyawaki et al., Proc. Natl. Acad. Sci. USA 96:2135-2140, 1999).
Oxidation-reduction (redox) processes are very important in living organisms. The formation of disulfide bonds during protein folding relies upon a well maintained redox buffering system of glutathione and oxidized glutathione (Carothers et al., Arch. Biochem. Biophys. 268:409425, 1989). There also exists a thioredoxin-like family of enzymes that catalyze the formation and isomerization of disulfide bonds in proteins (Debarbieux and Beckwith, Cell 99:117-119, 1999). In addition, redox signaling during apoptosis has been implicated in activating mitochondrial permeability transition, leading to cytochrome c release (Hall, Eur. J. Clin. Invest. 29:238-245, 1999). Redox changes in the form of cellular oxidation have also been suggested to be a final step in the apoptotic process leading to degradation of apoptotic bodies (Cai and Jones, J. Bioenerg. Biomemb. 31:327-334, 1999). Given the importance of in vivo processes such as protein folding and apoptosis that are dependant upon redox status, a non-invasive, convenient method for studying redox changes within living cells is needed.
Current methods of determining in vivo redox status have many limitations. Many present techniques require cells to be harvested before their contents can be analyzed. This type of procedure is not only very invasive but is also not a very accurate measure of the in vivo state of the cells. Moreover, it would be impossible with this technique to monitor redox changes within the same cell over a period of time. Recently, Keese et al. (Keese et al., FEBS Lett. 447:135-138, 1999) have developed an indicator of redox state in which glutathione reductase crystals were microinjected into the cytosol of human fibroblasts, and by detecting a color change of the crystals, they were able to determine the redox potential of the cytosol to be more reducing than −0.270 V. While this method may allow redox determination within single living cells, the cumbersome nature of the technique is still a major drawback. The most reasonable protocol for determining redox status is probably still that of Hwang et al. (Hwang et al., Science 257:1496-1502, 1992). They employed the tetrapeptide N-Acetyl-Asn-Tyr-Thr-Cys-NH2 to measure the ratio of thiol to disulfide in the cytosol and secretory pathway of cultured cells. They concluded that the cytosol is more reducing than the secretory pathway with an approximate redox potential of −0.221 to −0.236 V for the cytosol compared to −0.170 to −0.185 V for the secretory pathway. However, this method still required harvesting of the cells and like all the other methods, it is very labor intensive. Moreover, this technique determined redox potentials based only upon the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), potentially ignoring other redox buffering components.